PTF1a Activity in Enriched Posterior Foregut Endoderm, but Not Definitive Endoderm, Leads to Enhanced Pancreatic Differentiation in an In Vitro Mouse ESC-Based Model

Figure 1

Cell seeding density influences definitive endoderm generation. (a) Cultures seeded with two different mESC densities (100,000 cells/well or 300,000 cells/well of a 24-well plate) were subjected to a 3-day endoderm differentiation protocol containing 100 ng/mL bFGF, 100 ng/mL Activin A, and 10 ng/mL or 50 ng/mL of BMP4. (b) Cells cultured using another endoderm protocol containing Activin A 100 ng/mL or 30 ng/mL for 5 days in N2B27 but seeded with two starting cell densities (1000 cells/well or 5,000 cells/well of a 96 well plate). Relative mRNA expression of Sox17, T, and Foxa2 indicates low densities led to higher expression of DE markers. . Data are presented as mean ± SEM. Asterisks indicate values on comparison with corresponding high density cultures: , , and determined by one-way ANOVA with Tukey’s multiple comparison test. (c) Immunofluorescent costaining for FOXA2 and SOX17 confirms that cultures that started with low cell density (N2B27 A30 1000) had higher numbers of FOXA2⁺SOX17⁺ definitive endoderm than cultures seeded with high density (N2B27 A30 5000). Images at 5x. Higher magnification inset to indicate coexpression at the cellular level. Scale bars, 200 μm.

(a)

(b)

(c)